Figure 1. ULS1 deletion causes sensitivity to ACF due Top2 activity

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Figure 1. ULS1 deletion causes sensitivity to ACF due Top2 activity Figure 1. ULS1 deletion causes sensitivity to ACF due Top2 activity. (A) The 10-fold serial dilutions of WT (HFY9) or ... Figure 1. ULS1 deletion causes sensitivity to ACF due Top2 activity. (A) The 10-fold serial dilutions of WT (HFY9) or uls1Δ (HFY71) yeast on rich media (YPD) or drug containing plates (ACF). (B) Identification of isolated suppressor mutants and their location within the structure of the Top2 dimer (PDB ID: 4GFH). (C) Top2 point mutations were introduced into independent yeast strains to verify they are causing suppression. top2 I1121V (HFY264) and top2 Y510C (HFY263) alleles fully supress the ACF sensitivity of uls1Δ (HFY71) such that the grow identically to WT (HFY9) on ACF. (D) In vitro decatenation assay. A total of 200 nM of kinetoplastid DNA was incubated for 30 min at 30°C with 0, 3, 6, 12, 25, 50 or 100 nM Top2 before being run out on a 1% agarose gel. Top2 containing the suppressor mutation I1121V (HFP273) is ∼16-fold less active than wild-type Top2 (HFP 185) but still has significantly more activity than the ATPase dead Top2 E66Q (HFP271). A Coomassie-stained protein gel on the right illustrates the purity of expressed Top2 constructs. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz362, https://doi.org/10.1093/nar/gkz362 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. Physical interaction of Uls1 and Top2 is important for Uls1 function. (A) Yeast 2-hybrid assay. Yeast ... Figure 2. Physical interaction of Uls1 and Top2 is important for Uls1 function. (A) Yeast 2-hybrid assay. Yeast containing the indicated combination of Gal4 activator domain (pOAD) and Gal4 binding domain (pOBD) plasmids were grown on control (-LW) plates and assay (-LWH with 5 mM 3-Amino-1,2,4-triazole) plates. Full length Uls1 (HFP136) and Uls1 350–655 (HFP133) interact with Top2 (HFP 185) but not the empty vector control (HFP122). In contrast, Uls1 fragments 1–350 (HFP193) and 655–1619 (HFP134) do not bind Top2. (B) in vitro pulldown of full length Top2 with the indicated fragments of Uls1 bound to agarose beads showing input (I), flow-through (FT) and bound (B) fractions. (C) Diagram of Uls1 domain architecture. Serial dilutions of the indicated genotypes were assayed for viability on 250 μM ACF. Mutation of Uls1 ATPase function (uls1 E1109Q-HFY275) or deletion of its Top2 interaction domain (uls1 Δ350-655-HFY225) mimics uls1Δ (HFY71). In contrast, mutation of ULS1′s RING finger (uls1 C1385S-HFY230) has hardly any effect on ACF sensitivity whereas mutation of its five putative SIMs (HFY261) has a moderate effect on ACF sensitivity. (D) Western blot of the same constructs used in C, indicating equivalent expression levels. Ponceau-stained membrane is used a loading control. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz362, https://doi.org/10.1093/nar/gkz362 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. Uls1 has DNA-stimulated ATPase activity Figure 3. Uls1 has DNA-stimulated ATPase activity. (A) Scheme of the coupled ATPase assay used, reactions were carried ... Figure 3. Uls1 has DNA-stimulated ATPase activity. (A) Scheme of the coupled ATPase assay used, reactions were carried out at 30°C and A340 measurements taken every 10 s for 30 min. (B) ATP hydrolysis rates for the indicated proteins. The graph shows the average ± the standard deviation of three independent experiments. A total of 15 nM Uls1 was incubated with or without 100 μM salmon sperm DNA. (C) A Coomassie-stained protein gel on the right illustrates the purity of the purified Uls1 constructs Uls1 Δ1-349 (HFP385) and Uls1 Δ1-349, E1109Q (HFP404). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz362, https://doi.org/10.1093/nar/gkz362 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. Uls1 controls Top2 chromatin binding in the presence of ACF Figure 4. Uls1 controls Top2 chromatin binding in the presence of ACF. (A) Pairwise comparison of the average ChIP ... Figure 4. Uls1 controls Top2 chromatin binding in the presence of ACF. (A) Pairwise comparison of the average ChIP enrichment across all mapped reads (Genome) and specifically within common regions called as peaks by MACS2 (Peaks) in wild-type cells (HFY250) both in the presence or absence of 250 μM ACF. Top2 peaks become significantly more intense when ACF is added, Cohen's d = 0.49. (B) The same as in A, except in uls1Δ cells (HFY252) showing that the effect of ACF is exacerbated, Cohen's d = 1.56. (C) Pairwise comparison of the average ChIP enrichment in the presence of 250 μM ACF. Comparing common ACF-dependent peaks between wildtype (HFY250) and uls1Δ (HFY252) cells indicates that there is significantly more Top2 bound in uls1Δ, Cohen's d = 0.62. (D) Association of Top2 peaks within genes and the expression level of those genes in asynchronous culture under exponential growth. Expression data were taken from (45) and the number of peaks within each group is displayed next to the graph. (E) Normalized Top2 peak probability relative to the TSS of RNAP II transcripts in wild-type (HFY250) or uls1Δ (HFY252) cells in the presence or absence of ACF. The solid line displays the average with 95% confidence intervals indicated by the shaded area. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz362, https://doi.org/10.1093/nar/gkz362 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. Uls1 binding sites do not accumulate Top2 in the presence of ACF. (A) Pairwise comparison of the average Uls1 ... Figure 5. Uls1 binding sites do not accumulate Top2 in the presence of ACF. (A) Pairwise comparison of the average Uls1 ChIP enrichment (HFY176) across all mapped reads (Genome) and specifically within peak regions ± 100 μM ACF. The level of Uls1 chromatin binding is independent of ACF. (B) Normalized Uls1 peak probability relative to the TSS of RNA Pol II transcribed genes in the presence or absence of ACF. The solid line displays the average with 95% confidence intervals indicated by the shaded area. (C) Comparison of the average Top2 ChIP enrichment (using filtered reads) between regions that are either bound or unbound by Uls1 ± 250 μM ACF. In contrast to unbound sites, Uls1 binding sites do not accumulate Top2 in the presence of ACF. This effect is ULS1 dependent. (D) Pairwise comparison of the average Uls1 ChIP enrichment using unfiltered reads across the genome and specifically within tRNA genes ± 100 μM ACF. Uls1 becomes enriched at tRNA genes in the presence of ACF, Cohen's d = 1.16. (E) Same as (D) except looking at Top2 ChIP. ACF causes loss of Top2 from tRNA genes, which is ULS1 dependent. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz362, https://doi.org/10.1093/nar/gkz362 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 6. Model of how Uls1 and acriflavine influence Top2 DNA binding Figure 6. Model of how Uls1 and acriflavine influence Top2 DNA binding. (A) Summary of ChIP data describing how Uls1 ... Figure 6. Model of how Uls1 and acriflavine influence Top2 DNA binding. (A) Summary of ChIP data describing how Uls1 antagonizes the ACF-dependent increase in Top2 binding throughout the genome. (B) Model of how Uls1 might remodel a Top2 cleavage complex by promoting DNA-stimulated Top2 ATPase activity leading to movement of the transfer DNA (grey) and resolution of the Top2-DNA bonds within the guide DNA (black). Given that uls1Δ cells appear to have fewer Top2 peaks in the absence of ACF, it is possible that Uls1 may have an indirect role in promoting initial Top2 binding to DNA. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz362, https://doi.org/10.1093/nar/gkz362 The content of this slide may be subject to copyright: please see the slide notes for details.